title: A bidirectional promoter architecture enhances lentiviral transgenesis in embryonic and extraembryonic stem cells.
abstract: The two main challenges facing retroviral transgenesis are variable expression and epigenetic silencing. Although modern lentiviral vectors incorporate several elements to increase transgene expression and reduce position effect variegation and silencing, therapeutic research in stem cells, as well as production of transgenic animals, is still hampered by these two key problems. On the basis of recent studies demonstrating the chromatin insulating properties of divergent promoters, we sought to develop a bidirectional lentiviral vector with which to conduct RNA interference (RNAi)-based genetic screens in embryonic and extraembryonic stem cells. To this end, we designed and tested a series of synthetic bidirectional promoters, combining the mouse phosphoglycerate kinase 1 (Pgk1) promoter with other strong mammalian and viral promoters. Here, we demonstrate that a back-to-back configuration of the mouse Pgk1 and human eukaryotic translation elongation factor 1 alpha 1 promoters provided a substantive increase in both transgene expression and RNAi-based transcript depletion as compared with previous designs and other promoter combinations. Using this vector, we were able to achieve stable and robust depletion of a transfected luciferase reporter, as well as an endogenous non-coding RNA. The described constructs are an improved transgene delivery system capable of conducting RNAi screens in stem cells at single copy.
authors.affiliation: Department of Obstetrics and Gynecology, University of Western Ontario, Schulich School of Medicine and Dentistry, London, Ontario, Canada.
authors.forename: M C
authors.initials: MC
authors.lastname: Golding
authors.forename: M R W
authors.initials: MR
authors.lastname: Mann
mesh_terms: D000818:Animals; D002460:Cell Line; D053595:Embryonic Stem Cells; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D010735:Phosphoglycerate Kinase / Q000235:genetics; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D013234:Stem Cells
chemical_list: C094814:EEF1A1 protein, human; D020648:Peptide Elongation Factor 1; C528165:Pgk1 protein, mouse; D010735:Phosphoglycerate Kinase